Drug metabolome of the simvastatin formed by human intestinal microbiota in vitro

The human colon contains a diverse microbial population which contributes to degradation and metabolism of food components. Drug metabolism in the colon is generally poorly understood. Metabolomics techniques and in vitro colon models are now available which afford detailed characterization of drug metabolites in the context of colon metabolism. The aim of this work was to identify novel drug metabolites of Simvastatin (SV) by using an anaerobic human in vitro colon model at body temperature coupled with systems biology platform, excluding the metabolism of the host liver and intestinal epithelia. Comprehensive two-dimensional gas chromatography with a time-of-flight mass spectrometry (GC×GC-TOFMS) was used for the metabolomic analysis. Metabolites showing the most significant differences in the active faecal suspension were elucidated in reference with SV fragmentation and compared with controls: inactive suspension or buffer with SV, or with active suspension alone. Finally, time courses of selected metabolites were investigated. Our data suggest that SV is degraded by hydrolytic cleavage of methylbutanoic acid from the SV backbone. Metabolism involves demethylation of dimethylbutanoic acid, hydroxylation/dehydroxylation and β-oxidation resulting in the production of 2-hydroxyisovaleric acid (3-methyl-2-hydroxybutanoic acid), 3-hydroxybutanoic acid and lactic acid (2-hydroxypropanoic acid), and finally re-cyclisation of heptanoic acid (possibly de-esterified and cleaved methylpyranyl arm) to produce cyclohexanecarboxylic acid. Our study elucidates a pathway of colonic microbial metabolism of SV as well as demonstrates the applicability of the in vitro colon model and metabolomics to the discovery of novel drug metabolites from drug response profiles.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Problems Associated with Biological Markers of Alzheimer’s Disease

The etiopathogenesis of Alzheimer’s disease (AD) is still unclear, although clinical diagnostic criteria exist and the neuropathology of AD has been studied in great detail during the last 20 years. The present study addresses certain problems in the search for biological markers for the diagnosis, as well as in the follow-up of the course of AD and its differential diagnosis and reports some of our own observations in comparison with other studies. These include protein, genetic and neuroimaging markers. The definitions of biological markers and search strategies are also discussed. Special issue dedicated to Dr. Simo S. Oja     

Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.     

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.     

Effect of dietary supplementation with sea buckthorn (Hippophaë rhamnoides) seed and pulp oils on the fatty acid composition of skin glycerophospholipids of patients with atopic dermatitis

Sea buckthorn (Hippopha? rhamnoides) seed and pulp oils have traditionally been used for treating skin diseases in China and Russia, but are not widely used in other countries. A placebo-controlled, parallel study was carried out to investigate the effects of these oils on the fatty acid composition of skin glycerophospholipids of patients with atopic dermatitis. Sixteen patients ate 5 g of sea buckthorn seed oil, pulp oil, or paraffin oil daily for 4 months. Skin fatty acids were analyzed with gas chromatography before and after treatment. The seed oil slightly increased the proportion of docosapentaenoic acid (22:5n-3) and decreased the proportion of palmitic acid (16:0) in skin glycerophospholipids (0.05 < P < 0.1). The levels of the other fatty acids remained stable. The results show that the fatty acid composition of skin glycerophospholipids is well buffered against short-term dietary modification.     

Improved classification of Alzheimer's disease data via removal of nuisance variability

Diagnosis of Alzheimer's disease is based on the results of neuropsychological tests and available supporting biomarkers such as the results of imaging studies. The results of the tests and the values of biomarkers are dependent on the nuisance features, such as age and gender. In order to improve diagnostic power, the effects of the nuisance features have to be removed from the data. In this paper, four types of interactions between classification features and nuisance features were identified. Three methods were tested to remove these interactions from the classification data. In stratified analysis, a homogeneous subgroup was generated from a training set. Data correction method utilized linear regression model to remove the effects of nuisance features from data. The third method was a combination of these two methods. The methods were tested using all the baseline data from the Alzheimer's Disease Neuroimaging Initiative database in two classification studies: classifying control subjects from Alzheimer's disease patients and discriminating stable and progressive mild cognitive impairment subjects. The results show that both stratified analysis and data correction are able to statistically significantly improve the classification accuracy of several neuropsychological tests and imaging biomarkers. The improvements were especially large for the classification of stable and progressive mild cognitive impairment subjects, where the best improvements observed were 6% units. The data correction method gave better results for imaging biomarkers, whereas stratified analysis worked well with the neuropsychological tests. In conclusion, the study shows that the excess variability caused by nuisance features should be removed from the data to improve the classification accuracy, and therefore, the reliability of diagnosis making.     

cAMP mediates IL-1-induced lymphocyte penetration through endothelial monolayers

Endothelial cell incubated with IL-1 have been shown adhere more lymphocytes than nontreated endothelial cells. Here we demonstrate that IL-1 can also increase lymphocyte penetration through endothelial monolayers in vitro. IL-1 induced a transient increase in the number of lymphocytes penetrated through the endothelial monolayer into a filter in a time- and dose-dependent manner. This effect could be mimicked by increasing the cytosolic cAMP levels in the endothelial cells either by forskolin or dibutyryl-cAMP. Concomitantly we were able to show that IL-1 increased the cytosolic cAMP levels in endothelial cells. An inhibitor of adenylate cyclase, ddAdo, decreased both the IL-1-induced cAMP elevation and lymphocyte penetration. A protein kinase A inhibitor HA 1004 could inhibit the IL-1-induced lymphocyte penetration, where as protein kinase C (N-(2-guamidino-ethyl)-5-isoquinolinesyl foamide hydrocloride) and calcium-calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalensulfanamide) inhibitors had no effect. Adding dibutyryl-cGMP or calcium ionophore to the endothelial cells could not mimic IL-1-induced penetration and finally IL-1 did not induce PKC translocation in endothelial cells. These data support the view that IL-1 acts via cAMP as a second messenger in regard to lymphocyte penetration through endothelial cells. The above data demonstrate that IL-1-induced lymphocyte penetration through endothelial cells and that this IL-1-induced signal is transduced via cAMP in endothelial cells.     

A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth

Different dilutions of chlorhexidine gluconate were tested by the Kelsey-Sykes procedure. The method was further modified on microtitration plates using 2,3,5-triphenyltetrazolium chloride (TTC) reduction as an indicator of bacterial growth. There was a good correlation with the results based on TTC reduction and the conventional method based on turbidity changes caused by bacterial growth. Furthermore, the modified method using TTC reduction is more rapid and can be read by the naked eye because of the red colour.